Human pluripotent stem cells, such as human ES cells and human iPS cells, are receiving worldwide attention for their potential application to regenerative medicine. The requirement for the application of human pluripotent stem cells to regenerative medicine is to develop techniques for culturing and propagating such stem cells in a safe and stable manner. In particular, the development of a method for stably culturing such stem cells under the conditions in which no feeder cells are used and no xenogeneic components are contained in the culture system (xeno-free) is a pressing issue.
The present inventors found that early embryonic pluripotent stem cells utilize, as their scaffold, basement membranes containing laminin α5β1γ1 (laminin 511) as a major component, and first in the world, reported that a recombinant protein of human laminin 511 is a useful feeder-free culture matrix for human ES cells (see Non Patent Literature 1). In addition, the present inventors also reported that laminin 511 has very high affinity for α6β1 integrin and that a laminin 511E8 fragment (laminin 511E8) is comparable in α6β1 integrin binding activity to a full-length laminin 511 (see Non Patent Literature 2). Furthermore, the present inventors found that pluripotent stem cells in a dissociated single-cell state can be passaged on laminin 511E8, although such dissociated single-cell culture had been conventionally difficult, and reported that human laminin 511E8 is very effective as a feeder-free culture matrix for human pluripotent stem cells (see Patent Literature 1 and Non Patent Literature 3).
For cell culture using human laminin 511E8 as a matrix, the surface of a culture vessel should be coated with the human laminin 511E8 in advance. For human pluripotent stem cell culture, such coating is performed by adding a human laminin 511E8 solution onto a culture vessel at a final concentration of 0.25 to 2.0 μg/cm2 and incubating the culture vessel at 4° C. overnight or at room temperature up to 37° C. for 1 to 3 hours. However, laminins or their active fragments applied to coat the surface of the culture vessel are prone to inactivation by drying (see Non Patent Literature 4), and indeed, the present inventors confirmed that human laminin 511E8 applied to coat the surface of the culture vessel gradually became inactive after exposed to drying. Therefore, wider application of human laminin 511E8 as a culture matrix for human pluripotent stem cells in regenerative medicine etc. requires the development of a technology allowing the human laminin 511E8 applied as a coating component onto the surface of the culture vessel to be stably active in a dry state for a long period of time.